The synthesis of DNA is used for various purposes in the research field. Among them, much of the DNA syntheses are carried out by the enzymatic methods utilizing DNA polymerase, except for chemical synthesis of a short strand DNA such as an oligonucleotide. PCR, which can easily amplify the desired nucleic acid fragments in vitro, is highly valuable, and has become an indispensable tool in biology, medicine, agriculture, and other fields. In the amplification of DNA by PCR, high specificity of reaction is required in many cases. Despite developments of new DNA polymerase, and optimization of the composition of reaction mixture to reduce the non-specific amplification, there is still a need for improvements in PCR amplification specificity.
In addition to traditional PCR methods, which use DNA as the template material, DNA synthesis may also be achieved by utilizing the RNA-dependent DNA polymerase reverse transcriptase. In the research field, it is often necessary to analyze mRNA molecules derived from various genes. Reverse transcriptase enables the reverse transcription reaction for synthesizing cDNA using RNA as a template and, as a result, remarkable advancements have been achieved in the analysis of the mRNA molecules. The analysis of the mRNA molecules, in which the reverse transcriptase is used, is now an indispensable experimental technique in genetic studies, and has become an absolutely necessary technique in the various fields such as biology, medicine, and agriculture by applying in the cloning techniques and PCR techniques.
Some approaches have been so far made in order to improve the reactivity of the reverse transcription reaction. It has been studied, for example, a process of reverse transcription reaction which is performed at high temperatures; a cDNA synthesis process wherein a reverse transcriptase and an enzyme having the 3′-5′ exonuclease activity are used (JP Patent No. 3910015); a process wherein nucleic acid-binding proteins such as Ncp7, recA, SSB and T4gp32 are utilized (WO 00/55307); and the like. Despite the improvements that have been made in the reactivity of the reverse transcription reaction, cDNA of full-length may not be synthesized, and an amount of the synthesized cDNA may not be enough in some cases even today. Therefore, the improvement of the reactivity of the reverse transcription reaction is still an ongoing issue to be desirably further improved.
In addition to improvements to the two DNA synthesis methods described above, there is also a need in the scientific field for a technique by which to determine endoribonuclease cleavage-site specificity. mRNA interferases, for example, are sequence-specific endoribonucleases encoded by the toxin-antitoxin systems that are present in a wide range of bacterial genomes. These endoribonucleases usually have specific cleavage sites within single-strand RNA: E. coli MazF specifically cleaves at ACA sequences, ChpBK at ACY sequences (Y is U, A, or G), PemK from plasmid R100 at UAH sequences (where H is C, A, or U), and MazF-mt1 and MazF-mt6 from M. tuberculosis at UAC and U rich regions, respectively. It was recently found that a MazF homolog from Staphylococcus aureus cleaves at VUUV′ (where V and V′ are A, C, or G and may or may not be identical). However previous attempts to determine the cleavage specificities of some of the MazF homologs of M. tuberculosis have failed. Particularly for the determination of the cleavage specificity where a specific sequence longer than three bases is recognized, it is essential to use an RNA substrate that is long enough to cover all possible target sequences. A major problem in using long RNA as a substrate, however, is that although it is a single-stranded RNA, it forms extensive stable secondary structures. In order to use this or any other long RNA as a substrate for endoribonucleases, its secondary structures have to be unfolded. Thus there remains a need to develop new techniques to determine cleavage-site specificity for endoribonucleases such as mRNA interferases.
Cold shock proteins are found in various microorganisms, and are deemed to play some roles in the adaptation to the downshift of the growth temperature. Among them, CspA has been identified as a major cold shock protein. The gene encoding CspA was isolated from Escherichia coli, and a recombinant CspA was produced using its gene (see WO 90/09444). However, the conventionally proposed applications of cold shock proteins have been limited to their use as a cryoprotective protein which prevents freezing or frost damage in agricultural fields.
The present invention provides cold shock protein-containing compositions for improved DNA synthesis reactions with improved reactivity, methods for synthesizing DNA using such compositions, kits for use in such methods, and synthetic DNA products yielded by such methods. The present invention further provides cold shock protein-containing compositions for the identification of endoribonuclease cleavage sites, methods for identifying endoribonuclease cleavage sites using such compositions, and kits for use in such methods.